The invention relates to novel steroid derivatives and processes and intermediates for the preparation of these compounds.
Some compounds that associate strongly with the outer membrane of Gram-negative bacteria are known to disrupt the outer membrane and increase permeability. The increased permeability can increase the susceptibility of Gram-negative bacteria to other antibiotics. The best studied of this type of compound are the polymyxin antibiotics. For an example of a study involving the binding of polymyxin B to the primary constituent of the outer membrane of Gram-negative bacteria (lipid A) see: D. C. Morrison and D. M. Jacobs, Binding of Polymyxin B to The Lipid a Portion of Bacterial Lipopolysaccharides, Immunochemistry 1976, vol. 13, 813-819. For an example of a study involving the binding of a polymyxin derivative to Gram-negative bacteria see: M. Vaara and P. Viljanen, Binding of Polymyxin B Nonapeptide to Gram-negative Bacteria, Antimicrobial Agents and Chemotherapy, 1985, vol. 27, 548-554.
Membranes of Gram-negative bacteria are semipermeable molecular xe2x80x9csievesxe2x80x9d which restrict access of antibiotics and host defense molecules to their targets within the bacterial cell. Thus, cations and polycations which interact with and break down the outer membrane permeability barrier are capable of increasing the susceptibility of Gram-negative pathogenic bacteria to antibiotics and host defense molecules. Hancock and Wong demonstrated that a broad range of peptides could overcome the permeability barrier and coined the name xe2x80x9cpermeabilizersxe2x80x9d to describe them (Hancock and Wong, Antimicrob. Agents Chemother., 26:48, 1984).
The present invention features compounds of the formula I 
wherein:
fused rings A, B, C, and D are independently saturated or fully or partially unsaturated; and
each of R1 through R4, R6, R7, R11, R12, R15, R16, and R17 is independently selected from the group consisting of hydrogen, hydroxyl, a substituted or unsubstituted (C1-C10)alkyl, (C1-C10) hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl, (C1-C10) alkylcarboxy-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10) alkylamino, (C1-C10) alkylamino-(C1-C10) alkylamino-(C1-C10) alkylamino, a substituted or unsubstituted (C1-C10) aminoalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted arylamino-(C1-C10) alkyl, (C1-C10) haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo, a linking group attached to a second steroid, a substituted or unsubstituted (C1-C10) aminoalkyloxy, a substituted or unsubstituted (C1-C10) aminoalkyloxy-(C1-C10) alkyl, a substituted or unsubstituted (C1-C10) aminoalkylcarboxy, a substituted or unsubstituted (C1-C10) aminoalkylaminocarbonyl, a substituted or unsubstituted (C1-C10) aminoalkylcarboxamido, H2Nxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94Oxe2x80x94, H2Nxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94N(H)xe2x80x94, (C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HNxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94Oxe2x80x94, (C1-C10) guanidinoalkyl oxy, (C1-C10) quaternaryammoniumalkylcarboxy, and (C1-C10) guanidinoalkyl carboxy, where Q5 is a side chain of any amino acid (including the side chain of glycine, i.e., H), P.G. is an amino protecting group, and
R5, R8, R9, R10, R13, and R14 is each independently: deleted when one of fused rings A, B, C, or D is unsaturated so as to complete the valency of the carbon atom at that site, or
selected from the group consisting of hydrogen, hydroxyl, a substituted or unsubstituted (C1-C10) alkyl, (C1-C10) hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl, a substituted or unsubstituted (C1-C10) aminoalkyl, a substituted or unsubstituted aryl, C1-C10 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, a linking group attached to a second steroid, a substituted or unsubstituted (C1-C10) aminoalkyloxy, a substituted or unsubstituted (C1-C10) aminoalkylcarboxy, a substituted or unsubstituted (C1-C10) aminoalkylaminocarbonyl, H2Nxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94Oxe2x80x94, H2Nxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94N(H)xe2x80x94, (C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HNxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94Oxe2x80x94, (C1-C10) guanidinoalkyloxy, and (C1-C10) guanidinoalkylcarboxy, where Q5 is a side chain of any amino acid, P.G. is an amino protecting group, and
provided that at least two of R1 through R14 are independently selected from the group consisting of a substituted or unsubstituted (C1-C10) aminoalkyloxy, (C1-C10) alkylcarboxy-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10) alkylamino, (C1-C10) alkylamino-(C1-C10) alkylamino-(C1-C10) alkylamino, a substituted or unsubstituted (C1-C10) aminoalkylcarboxy, a substituted or unsubstituted arylamino-(C1-C10) alkyl, a substituted or unsubstituted (C1-C10) aminoalkyloxy-(C1-C10) alkyl, a substituted or unsubstituted (C1-C10) aminoalkylaminocarbonyl, (C1-C10) quaternaryammonium alkylcarboxy, H2Nxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94Oxe2x80x94, H2Nxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94N(H)xe2x80x94, (C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HNxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94Oxe2x80x94, (C1-C10) guanidinoalkyloxy, and (C1-C10) guanidinoalkylcarboxy; or a pharmaceutically acceptable salt thereof.
The term fused ring used herein can be heterocyclic or carbocyclic, preferably.
The term xe2x80x9csaturatedxe2x80x9d used herein refers to the fused ring of formula I having each atom in the fused ring either hydrogenated or substituted such that the valency of each atom is filled.
The term xe2x80x9cunsaturatedxe2x80x9d used herein refers to the fused ring of formula I where the valency of each atom of the fused ring may not be filled with hydrogen or other substituents. For example, adjacent carbon atoms in the fused ring can be doubly bound to each other. Unsaturation can also include deleting at least one of the following pairs and completing the valency of the ring carbon atoms at these deleted positions with a double bond; such as R5 and R9; R8 and R10; and R13 and R14.
The term xe2x80x9cunsubstitutedxe2x80x9d used herein refers to a moiety having each atom hydrogenated such that the valency of each atom is filled.
The term xe2x80x9chaloxe2x80x9d used herein refers to a halogen atom such as fluorine, chlorine, bromine, or iodine.
Examples of amino acid side chains include but are not limited to H (glycine), methyl (alanine), xe2x80x94CH2xe2x80x94(Cxe2x95x90O)xe2x80x94NH2 (asparagine), xe2x80x94CH2xe2x80x94SH (cysteine), and xe2x80x94CH(OH)CH3 (threonine).
An alkyl group is a branched or unbranched hydrocarbon that may be substituted or unsubstituted. Examples of branched alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, sec-pentyl, isopentyl, tert-pentyl, isohexyl. Substituted alkyl groups may have one, two, three or more substituents, which may be the same or different, each replacing a hydrogen atom. Substituents are halogen (e.g., F, Cl, Br, and I), hydroxyl, protected hydroxyl, amino, protected amino, carboxy, protected carboxy, cyano, methylsulfonylamino, alkoxy, acyloxy, nitro, and lower haloalkyl.
The term xe2x80x9csubstituedxe2x80x9d used herein refers to moieties having one, two, three or more substituents, which may be the same or different, each replacing a hydrogen atom. Examples of substituents include but are not limited to halogen (e.g., F, Cl, Br, and I), hydroxyl, protected hydroxyl, amino, protected amino, carboxy, protected carboxy, cyano, methylsulfonylamino, alkoxy, alkyl, aryl, aralkyl, acyloxy, nitro, and lower haloalkyl.
An aryl group is a C6-20 aromatic ring, wherein the ring is made of carbon atoms (e.g., C6-14, C6-10 aryl groups). Examples of haloalkyl include fluoromethyl, dichloromethyl, trifluoromethyl, 1,1-difluoroethyl, and 2,2-dibromoethyl.
An aralkyl group is a group containing 6-20 carbon atoms that has at least one aryl ring and at least one alkyl or alkylene chain connected to that ring. An example of an aralkyl group is a benzyl group.
A linking group is any divalent moiety used to link a compound of formula to another steroid, e.g., a second compound of formula I. An example of a linking group is (C1-C10) alkyloxy-(C1-C10) alkyl.
Numerous amino-protecting groups are well-known to those in the art. In general, the species of protecting group is not critical, provided that it is stable to the conditions of any subsequent reaction(s) on other positions of the compound and can be removed at the appropriate point without adversely affecting the remainder of the molecule. In addition, a protecting group may be substituted for another after substantive synthetic transformations are complete. Clearly, where a compound differs from a compound disclosed herein only in that one or more protecting groups of the disclosed compound has been substituted with a different protecting group, that compound is within the invention. Further examples and conditions are found in T. W. Greene, Protective Groups in Organic Chemistry, (1st ed., 1981, 2nd ed., 1991).
The present invention also includes methods of synthesizing compounds of formula I where at least two of R1 through R14 are independently selected from the group consisting of a substituted or unsubstituted (C1-C10) aminoalkyloxy. The method includes the step of contacting a compound of formula IV, 
where at least two of R1 through R14 are hydroxyl, and the remaining moieties on the fused rings A, B, C, and D are defined for formula I, with an electrophile to produce an alkyl ether compound of formula IV, wherein at least two of R1 through R14 are (C1-C10)alkyloxy. The alkyl ether compounds are converted into an amino precursor compound wherein at least two of R1 through R14 are independently selected from the group consisting of (C1-C10) azidoalkyloxy and (C1-C10) cyanoalkyloxy and the amino precursor compound is reduced to form a compound of formula I.
The electrophiles used in the method include but are not limited to 2-(2-bromoethyl)-1,3-dioxolane, 2-iodoacetamide, 2-chloroacetamide, N-(2-bromoethyl)phthalimide, N-(3-bromopropyl)phthalimide, and allybromide. The preferred electrophile is allylbromide.
The invention also includes a method of producing a compound of formula I where at least two of R1 through R14 are (C1-C10) guanidoalkyloxy. The method includes contacting a compound of formula IV, where at least two of R1 through R14 are hydroxyl, with an electrophile to produce an alkyl ether compound of formula IV, where at least two of R1 through R14 are (C1-C10)alkyloxy. The allyl ether compound is converted into an amino precursor compound where at least two of R1 through R14 are independently selected from the group consisting of (C1-C10) azidoalkyloxy and (C1-C10) cyanoalkyloxy. The amino precursor compound is reduced to produce an aminoalkyl ether compound wherein at least two of R1 through R14 are (C1-C10) aminoalkyloxy. The aminoalkyl ether compound is contacted with a guanidino producing electrophile to form a compound of formula I.
The term xe2x80x9cguanidino producing electrophilexe2x80x9d used herein refers to an electrophile used to produce a guanidino compound of formula I. An example of an guanidino producing electrophile is HSO3xe2x80x94C(NH)xe2x80x94NH2.
The invention also includes a method of producing a compound of formula I where at least two of R1 through R14 are H2Nxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94Oxe2x80x94 and Q5 is the side chain of any amino acid. The method includes the step of contacting a compound of formula IV, where at least two of R1 through R14 are hydroxyl, with a protected amino acid to produce a protected amino acid compound of formula IV where at least two of at least two of R1 through R14 are P.G.-HNxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94Oxe2x80x94 and Q5 is the side chain of any amino acid and P.G. is an amino protecting group. The protecting group of the protected amino acid compound is removed to form a compound of formula I.
The present invention also includes pharmaceutical compositions of matter that are useful as antibacterial agents, sensitizers of bacteria to other antibiotics and disrupters of bacterial membranes. The pharmaceutical compositions can be used to treat humans and animals having a bacterial infection. The pharmaceutical compositions can include an effective amount of the steroid derivative alone or in combination with other antibacterial agents.
The invention further includes a method of preparing the compound (A): 
by
(a) contacting 5xcex2-cholanic acid 3,7,12-trione methyl ester with hydroxyl amine hydrochloride and sodium acetate to form the trioxime (B): 
and
(b) contacting trioxime (B) with NaBH4 and TiCl4 to yield compound (A).
The invention also includes a compound comprising a ring system of at least 4 fused rings, where each of the rings has from 5-7 atoms. The ring system has two faces, and contains 3 chains attached to the same face. Each of the chains contains a nitrogen-containing group that is separated from the ring system by at least one atom; the nitrogen-containing group is an amino group, e.g., a primary amino group, or a guanidino group. Preferably, the compound also contains a hydrophobic group, such as a substituted (C3-10) aminoalkyl group, a (C1-10) alkyloxy (C3-10) alkyl group, or a (C1-10) alkylamino (C3-10)alkyl group, attached to the steroid backbone.
For example, the compound may have the formula V, where each of the three chains containing nitrogen-containing groups is independently selected from R1 through R4, R6, R7, R11, R12, R15, R16, R17, and R18, defined below. 
where:
each of fused rings A, B, C, and D is independently saturated, or is fully or partially unsaturated, provided that at least two of A, B, C, and D are saturated, wherein rings A, B, C, and D form a ring system;
each of m, n, p, and q is independently 0 or 1;
each of R1 through R4, R6, R7, R11, R12, R15, R16, R17, and R18 is independently selected from the group consisting of hydrogen, hydroxyl, a substituted or unsubstituted (C1-C10) alkyl, (C1-C10) hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl, (C1-C10) alkylcarboxy-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10) alkylamino, (C1-C10) alkylamino-(C1-C10) alkylamino-(C1-C10) alkylamino, a substituted or unsubstituted (C1-C10) aminoalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted arylamino-(C1-C10) alkyl, (C1-C10) haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo, a linking group attached to a second steroid, a substituted or unsubstituted (C1-C10) aminoalkyloxy, a substituted or unsubstituted (C1-C10) aminoalkyloxy-(C1-C10) alkyl, a substituted or unsubstituted (C1-C10) aminoalkylcarboxy, a substituted or unsubstituted (C1-C10) aminoalkylaminocarbonyl, a substituted or unsubstituted (C1-C10) aminoalkylcarboxamido, H2Nxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94Oxe2x80x94, H2Nxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94N(H)xe2x80x94, (C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HNxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94Oxe2x80x94, (C1-C10) guanidinoalkyl oxy, (C1-C10) quaternaryammoniumalkylcarboxy, and (C1-10) guanidinoalkyl carboxy, where Q5 is a side chain of any amino acid (including the side chain of glycine, i.e., H), P.G. is an amino protecting group; and
each of R5, R8, R9, R10, R13, and R14 is independently: deleted when one of fused rings A, B, C, or D is unsaturated so as to complete the valency of the carbon atom at that site, or selected from the group consisting of hydrogen, hydroxyl, a substituted or unsubstituted (C1-C10) alkyl, (C1-C10) hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl, a substituted or unsubstituted (C1-C10) aminoalkyl, a substituted or unsubstituted aryl, C1-C10 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, a linking group attached to a second steroid, a substituted or unsubstituted (C1-C10) aminoalkyloxy, a substituted or unsubstituted (C1-C10) aminoalkylcarboxy, a substituted or unsubstituted (C1-C10) aminoalkylaminocarbonyl, H2Nxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94Oxe2x80x94, H2Nxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94N(H)xe2x80x94, (C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HNxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94Oxe2x80x94, (C1-C10) guanidinoalkyloxy, and (C1-C10) guanidinoalkylcarboxy, where Q5 is a side chain of any amino acid, P.G. is an amino protecting group,
provided that at least three of R1 through R4, R6, R7, R11, R12, R15, R16, R17, and R18 are disposed on the same face of the ring system and are independently selected from the group consisting of a substituted or unsubstituted (C1-C10) aminoalkyloxy, (C1-C10) alkylcarboxy-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10) alkylamino, (C1-C10) alkylamino-(C1-C10) alkylamino-(C1-C10) alkylamino, a substituted or unsubstituted (C1-C10) aminoalkylcarboxy, a substituted or unsubstituted arylamino-(C1-C10) alkyl, a substituted or unsubstituted (C1-C10) aminoalkyloxy-(C1-C10) alkyl, a substituted or unsubstituted (C1-C10) aminoalkylaminocarbonyl, (C1-C10) quaternaryammoniumalkylcarboxy, H2Nxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94Oxe2x80x94, H2Nxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94N(H)xe2x80x94, (C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HNxe2x80x94HC(Q5)xe2x80x94C(O)xe2x80x94Oxe2x80x94, (C1-C10) guanidinoalkyloxy, and (C1-C10) guanidinoalkylcarboxy; or a pharmaceutically acceptable salt thereof. Preferably, at least two, or at least, three, of m, n, p, and q are 1.
Without wishing to be bound to any particular theory, the steroid derivatives described herein act as bacteriostatic and bactericidal agents by binding to the outer cellular membrane of bacteria. The interaction between the steroid derivatives and the bacteria membrane disrupts the integrity of the cellular membrane and results in the death of the bacteria cell. In addition, compounds of the present invention also act to sensitize bacteria to other antibiotics. At concentrations of the steroid derivatives below the corresponding minimum bacteriostatic concentration, the derivatives cause bacteria to become more susceptible to other antibiotics by increasing the permeability of the outer membrane of the bacteria. Measurements used to quantitate the effects of the steroid derivatives on bacteria include: measurement of minimum inhibitory concentrations (MICs), measurement of minimum bactericidal concentrations (MBCs) and the ability of the steroid derivatives to lower the MICs of other antibiotics, e.g., erythromycin and novobiocin.
A person of skill will recognize that the compounds described herein preserve certain stereochemical and electronic characteristics found in steroids. The term xe2x80x9csame configurationxe2x80x9d as used herein refers to substituents on the fused steroid having the same stereochemical orientation. For example substituents R3, R7 and R12 are all xcex2-substituted or xcex1-substituted. The configuration of the moieties R3, R7, and R12 substituted on C3, C7, and C12 may be important for interaction with the cellular membrane.
In another aspect, the invention features several methods of using the above-described compounds. For example, an effective amount of an anti-microbial composition comprising such a compound is administered to a host (including a human host) to treat a microbial infection. The compound by itself may provide the anti-microbial effect, in which case the amount of the compound administered is sufficient to be anti-microbial. Alternatively, an additional anti-microbial substance to be delivered to the microbial cells (e.g., an antibiotic) is included in the anti-microbial composition. By facilitating delivery to the target cells, the compounds can enhance the effectiveness of the additional antimicrobial substance. In some cases the enhancement may be substantial. Particularly important target microbes are bacteria (e.g., Gram-negative bacteria generally or bacteria which have a substantial ( greater than 40%) amount of a lipid A or lipid A-like substance in the outer membrane). Other microbes including fungi, viruses, and yeast may also be the target organisms.
The compounds can also be administered in other contexts to enhance cell permeability to introduce any of a large number of different kinds of substances into a cell, particularly the bacterial cells discussed above. In addition to introducing anti-microbial substances, the invention may be used to introduce other substances such as macromolecules (e.g., vector-less DNA).
The invention can also be used to make anti-microbial compositions (e.g., disinfectants, antiseptics, antibiotics etc.) which comprise one of the above compounds. These compositions are not limited to pharmaceuticals, and they may be used topically or in non-therapeutic contexts to control microbial (particularly bacterial) growth. For example, they may be used in applications that kill or control microbes on contact.
In yet another aspect, the invention generally features methods of identifying compounds that are effective against a microbe by administering a candidate compound and a compound according to the invention the microbe and determining whether the candidate compound has a static or toxic effect (e.g, an antiseptic, germicidal, disinfectant, or antibiotic effect) on the microbe. Again, bacteria such as those discussed above are preferred. This aspect of the invention permits useful testing of an extremely broad range of candidate anti-microbials which are known to have anti-microbial effect in some contexts, but which have not yet been shown to have any effect against certain classes of microbes such as the bacteria discussed above. As described in greater detail below, this aspect of the invention permits testing of a broad range of antibiotics currently thought to be ineffective against Gram-negative or lipid A-like containing bacteria.
In yet another aspect the invention features compositions which include one of the above compounds in combination with a substance to be introduced into a cell such as an antimicrobial substance as described in greater detail above. The compound and the additional substance may be mixed with a pharmaceutically acceptable carrier.
Other features or advantages of the present invention will be apparent from the following detailed description of several embodiments, and also from the appending claims.
The invention encompasses steroid derivatives that can be made by the synthetic routes described herein, and methods of treating a subject having a condition mediated by a bacterial infection by administering an effective amount of a pharmaceutical composition containing a compound disclosed herein to the subject.